Haloarcula mannanilytica sp. nov., a galactomannan-degrading haloarchaeon isolated from commercial salt.

A mannan-degrading halophilic archaeal strain, MD130-1T, was isolated from a commercial salt sample. Cells were motile, rod-shaped, and stained Gram-negative. Colonies were pink pigmented. Strain MD130-1T was able to grow at 1.5-4.6 M NaCl (optimum, 3.6 M) at pH 6.0-8.0 (optimum, pH 7.0) and at 25-50 °C (optimum, 40 °C). The DNA G+C content was 62.1 mol% (genome). The orthologous 16S rRNA gene sequence showed the highest similarity (99.4 %) to those of Haloarcula japonica JCM 7785T and Haloarcula hispanica JCM 8911T. The values of genome relatedness between strain MD130-1T and Haloarcula species were 84.33-85.96 % in ANIb and 30.4-32.9 % using GGDC formula 2. The polar lipids of strain MD130-1T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and triglycosyl diether-2. Based on the results of phenotypic and phylogenetic analyses, the strain represents a new species of the genus Haloarcula, for which the name Haloarcula mannanilytica sp. nov. is proposed. The type strain is MD130-1T (=JCM 33835T=KCTC 4287T) isolated from commercial salt made in Ishikawa prefecture, Japan.

Discovery of bacteriorhodopsins in Haloarchaeal species isolated from Indian solar salterns: deciphering the role of the N-terminal residues in protein folding and functional expression.

Interesting optical and photochemical properties make microbial rhodopsin a promising biological material suitable for various applications, but the cost-prohibitive nature of production has limited its commercialization. The aim of this study was to explore the natural biodiversity of Indian solar salterns to isolate natural bacteriorhodopsin (BR) variants that can be functionally expressed in Escherichia coli. In this study, we report the isolation, functional expression and purification of BRs from three pigmented haloarchaea, wsp3 (water sample Pondicherry), wsp5 and K1T isolated from two Indian solar salterns. The results of the 16S rRNA data analysis suggest that wsp3, wsp5 and K1T are novel strains belonging to the genera Halogeometricum, Haloferax and Haloarcula respectively. Overall, the results of our study suggest that 17 N-terminal residues, that were not included in the gene annotation of the close sequence homologues, are essential for functional expression of BRs. The primary sequence, secondary structural content, thermal stability and absorbance spectral properties of these recombinant BRs are similar to those of the previously reported Haloarcula marismortui HmBRI. This study demonstrates the cost-effective, functional expression of BRs isolated from haloarchaeal species using E. coli as an expression host and paves the way for feasibility studies for future applications.

Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment.

A halophilic organism, SWO25T, was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25T grew optimally at 35-45 °C, at pH 6.0-8.0 and 0.05-0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3-5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25T to the genus Haloarcula. The DNA G+C content was 61.1mol%. DNA-DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB' genes showed that strain SWO25T is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarculasebkhae sp. nov. is proposed. The type strain is SWO25T (=CIP 110583T=JCM 19018T).

Temperature-dependent expression of different guanine-plus-cytosine content 16S rRNA genes in Haloarcula strains of the class Halobacteria.

Haloarcula strains, which are halophilic archaea, harbour two to three copies of 16S rRNA genes (rrsA, rrsB and rrsC) in their genomes. While rrsB and rrsC (rrsBC) show almost identical sequences, rrsA shows 4-6% sequence difference and 1-3% guanine-plus-cytosine content (PGC) difference compared to rrsBC. Based on the strong correlation between the PGC of 16S rRNA genes and the growth temperatures of the prokaryotes, we hypothesised that high-PGCrrsA and low-PGCrrsBC are expressed at high and low temperatures, respectively. To verify the hypothesis, we performed sequence analyses and expression surveys of each 16S rRNA gene in eight Haloarcula strains. The secondary structure prediction of the 16S rRNA via computer simulation showed that the structural stability of 16S rRNAs transcribed from rrsA was higher than that of 16S rRNAs transcribed from rrsBC. We measured expression levels of rrsA and rrsBC under various temperature conditions by reverse-transcriptase quantitative PCR. The expression ratio of high-PGCrrsA to low-PGCrrsBC increased with cultivation temperatures in seven of eight Haloarcula strains. Our results suggest that the transcription of high-PGCrrsA and low-PGCrrsBC may be regulated in response to environmental temperature, and that 16S rRNAs transcribed from high-PGCrrsA function under high temperature conditions close to the maximum growth temperature.

Characterization of an organic solvent-tolerant lipase from Haloarcula sp. G41 and its application for biodiesel production.

A haloarchaeal strain G41 showing lipolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterizations along with 16S rRNA gene sequence analysis placed the isolate in the genus Haloarcula. Lipase production was strongly influenced by the salinity of growth medium with maximum in the presence of 20% NaCl or 15% Na2SO4. The lipase was purified to homogeneity with a molecular mass of 45 kDa. Substrate specificity test revealed that it preferred long-chain p-nitrophenyl esters. The lipase was highly active and stable over broad ranges of temperature (30-80 °C), pH (6.0-11.0), and NaCl concentration (10-25%), with an optimum at 70 °C, pH 8.0, and 15% NaCl, showing thermostable, alkali-stable, and halostable properties. Enzyme inhibition studies indicated that the lipase was a metalloenzyme, with serine and cysteine residues essential for enzyme function. Moreover, it displayed high stability and activation in the presence of hydrophobic organic solvents with log Pow ≥ 2.73. The free and immobilized lipases from strain G41 were applied for biodiesel production, and 80.5 and 89.2% of yields were achieved, respectively. This study demonstrated the feasibility of using lipases from halophilic archaea for biodiesel production.

Halostable cellulase with organic solvent tolerance from Haloarcula sp. LLSG7 and its application in bioethanol fermentation using agricultural wastes.

A haloarchaeal strain LLSG7 with cellulolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterization along with 16S rRNA gene sequence analysis placed the isolate in the genus Haloarcula. Cellulase production was strongly influenced by the salinity of the culture medium with the maximum obtained in the presence of 25 % NaCl. Substrate specificity tests showed that the crude cellulase was a multicomponent enzyme system, and zymogram analysis revealed that five different endoglucanases were secreted by strain LLSG7. Optimal cellulase activity was at 50 °C, pH 8.0, and 20 % NaCl. In addition, it was highly active and stable over broad ranges of temperature (40-80 °C), pH (7.0-11.0), and NaCl concentration (17.5-30 %). The cellulase displayed remarkable stability in the presence of non-polar organic solvents with log P ow ≥ 1.97. The crude cellulase secreted by strain LLSG7 was further applied to hydrolyze alkali-pretreated rice straw and the enzymatic hydrolysate was used as the substrate for bioethanol fermentation by Saccharomyces cerevisiae. The yield of ethanol was 0.177 g per gram of pretreated rice straw, suggesting that it might be potentially useful for bioethanol production.

Haloarcula salaria sp. nov. and Haloarcula tradensis sp. nov., isolated from salt in Thai fish sauce.

Two red-pigmented, strictly aerobic, pleomorphic rod-shaped and extremely halophilic archaea, designated strains HST01-2R(T) and HST03(T), were isolated from salt in a fish sauce sample from Thailand. The novel strains grew optimally at 37 °C, pH 7.0, and in the presence of 20-25 % (w/v) NaCl. The DNA G+C contents of the isolates were 61.6-62.2 mol%. Phylogenetic analysis based on a comparison of 16S rRNA gene sequences revealed that strains HST01-2R(T) and HST03(T) were placed in the radiation of species of the genus Haloarcula. The chemotaxonomic properties of the two strains, i.e. the presence of MK-8 and MK-8(H(2)) as the major menaquinone components and C(20)C(20) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a triglycosyl diether as major polar lipids, supported the assignment of the two strains to the genus Haloarcula. Nevertheless, several phenotypic features and the low DNA-DNA relatedness between the two strains and related species of the genus Haloarcula (13.4-46.9 %) enabled the strains to be distinguished from each other and from recognized species. Therefore, strains HST01-2R(T) and HST03(T) represent two novel species in the genus Haloarcula, for which the names Haloarcula salaria sp. nov. and Haloarcula tradensis sp. nov. are proposed, respectively. The type strains are HST01-2R(T) (=BCC 40029(T)=JCM 15759(T)=PCU 313(T)) and HST03(T) (=BCC 40030(T)=JCM 15760(T)=PCU 314(T)).

Isolation of hydrocarbon-degrading extremely halophilic archaea from an uncontaminated hypersaline pond (Camargue, France).

Little information exists about the ability of halophilic archaea present in hypersaline environments to degrade hydrocarbons. In order to identify the potential actors of hydrocarbon degradation in these environments, enrichment cultures were prepared using samples collected from a shallow crystallizer pond with no known contamination history in Camargue, France, with n-alkanes provided as source of carbon and energy. Five alkane-degrading halophilic archaeal strains were isolated: one (strain MSNC 2) was closely related to Haloarcula and three (strains MSNC 4, MSNC 14, and MSNC 16) to Haloferax. Biodegradation assays showed that depending on the strain, 32 to 95% (0.5 g/l) of heptadecane was degraded after 30 days of incubation at 40 degrees C in 225 g/l NaCl artificial medium. One of the strains (MSNC 14) was also able to degrade phenanthrene. This work clearly shows for the first time the potential role of halophilic archaea belonging to the genera Haloarcula and Haloferax in the degradation of hydrocarbons in both pristine and hydrocarbon-contaminated hypersaline environments.

Haloarcula amylolytica sp. nov., an extremely halophilic archaeon isolated from Aibi salt lake in Xin-Jiang, China.

A starch-hydrolysing and extremely halophilic archaeon (strain BD-3(T)), isolated from Aibi salt lake in Xin-Jiang, China, was characterized phenotypically and genotypically in order to determine its taxonomic status. On the basis of its polar lipid composition, nucleotide sequences of its 16S rRNA genes, genomic DNA G+C content (62.4 mol%) and growth characteristics, the organism could be assigned to the genus Haloarcula. Phenotypic differences and low DNA-DNA hybridization values to related Haloarcula species distinguished strain BD-3(T) from recognized Haloarcula species. It is therefore concluded that strain BD-3(T) represents a novel species, for which the name Haloarcula amylolytica sp. nov. is proposed. The type strain is BD-3(T) (=CGMCC 1.5335(T)=JCM 13557(T)).

Halosimplex carlsbadense gen. nov., sp. nov., a unique halophilic archaeon, with three 16S rRNA genes, that grows only in defined medium with glycerol and acetate or pyruvate.

A halophilic archaeon has been isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico. This microorganism grows only on defined media supplemented with either a combination of acetate and glycerol, glycerol and pyruvate, or pyruvate alone. The archaeon is unable to grow on complex media or to use carbohydrates, amino acids, fats, proteins, or nucleic acids for growth. Unlike other halophilic microbes, this organism possesses four glycolipids, two of which may be novel. The microbe is unique in that it has three dissimilar 16S rRNA genes. Two of the three genes show only 97% similarity to one another, while the third gene possesses only 92%-93% similarity to the other two. Inferred phylogenies indicate that the organism belongs to a deep branch in the line of Haloarcula and Halorhabdus. All three lines of taxonomic evidence: phenotype, lipid patterns, and phylogeny, support creation of a new genus and species within the halophilic Archaea. The name suggested for this new genus and species is Halosimplex carlsbadense. The type strain is 2-9-1(T) (= ATCC BAA-75 and JCM 11222) as written in the formal description.

Taxonomic characterization of Haloferax sp. (" H. alicantei") strain Aa 2.2: description of Haloferax lucentensis sp. nov.

An extremely halophilic archaeon, previously named as Haloferax sp. strain Aa 2.2 or "Haloferax alicantei" that has been extensively used for genetic studies with halobacteria, was taxonomically characterized by using phenotypic tests (including morphological, physiological, biochemical and nutritional features), DNA-DNA hybridization and 16S rRNA sequence phylogenetic analysis. This organism was isolated in 1986 by Torreblanca et al. from a pond of a Spanish saltern located in Alicante. The cells were pleomorphic, Gram negative and grew optimally at 25% NaCl. The polar lipid composition was similar to that of species of the genus Haloferax. The DNA G+C content of this strain was 64.5 mol%. Phylogenetic analysis based on 16S rRNA sequence comparison confirmed that this archaeon is a member of the genus Haloferax and was most closely related to Haloferax volcanii. DNA-DNA hybridization between strain Aa 2.2 and the type strain of all named species of the genus Haloferax revealed low levels of relatedness (25-2%), supporting the placement of this organism in a new species. On the basis of the phenotypic characteristics, molecular data and phylogenetic analysis we propose to name strain Aa 2.2 as a new species, Haloferax lucentensis sp. nov. The type strain is Aa 2.2 (=JCM 9276=NCIMB 13854=CIP 107410=DSM 14919=CECT 5871=CCM 7023).

Taxonomic analysis of extremely halophilic archaea isolated from 56-years-old dead sea brine samples.

A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the S(J) coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et al., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work.

Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt).

The motile, predominantly square-shaped, red archaeon strain 801030/1T, isolated from a brine pool in the Sinai peninsula (Egypt), was characterized taxonomically. On the basis of its polar lipid composition, the nucleotide sequences of its two 16S rRNA genes, the DNA G+C content (60.1 mol%) and its growth characteristics, the isolate could be assigned to the genus Haloarcula. However, phylogenetic analysis of the two 16S rRNA genes detected in this organism and low DNA-DNA hybridization values with related Haloarcula species showed that strain 801030/1T is sufficiently different from the recognized Haloarcula species to warrant its designation as a new species. A new species, Haloarcula quadrata, is therefore proposed, with strain 801030/1T (= DSM 11927T) as the type strain.